This invention relates to detecting bacteria belonging to the genus Salmonella. (The term "Salmonella," as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology, infra). Detection of Salmonella bacteria is important in various medical and public health contexts. From the standpoint of human disease, Salmonella species are one of the most important bacteria. Salmonella bacteria can cause a variety of infections ranging from simple gastroenteritis to more severe illnesses.
According to a standard laboratory method, the presence of Salmonella in clinical specimens (e.g., stool) is detected by culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms. The resulting colonies are examined for morphological and biochemical characteristics, a process that typically is started 48 hours after acquisition of the sample and takes several days to complete.
Taber et al., U.S. Pat. No. 4,689,295, discloses the use of DNA probes specific for Salmonella DNA to detect the presence of bacteria of the genus Salmonella in food.
Kohne et al. (1968), Biophysical Journal 8: 1104-1118, discuss one method for preparing probes to rRNA sequences.
Pace and Campbell, (1971), Journal of Bacteriology 107: 543-547, discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating these homology levels.
Sogin, et al. (1972), Journal of Molecular Evolution 1: 173-184, discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships.
Fox et al. (1977), International Journal of Systematic Bacteriology 27: 44-57, discuss the comparative cataloging (of 16S ribosomal RNAs) approach to prokaryotic systematics.
Kohne et al., (1983), Gen-Probe Patent Appl., (Ref.?) describe a strategy for obtaining nucleic acid fragments for use as probes to ribosomal RNA molecules.
The present invention will be better understood in light of the following definitions:
DNA--an abbreviation for deoxyribonucleic acid, the type of nucleic acid containing deoxyribose as the sugar component and considered to be the repository of hereditary charcteristics; i.e., the genetic material of which genes are composed.
RNA--an abbreviation for ribonucleic acid, the type of nucleic acid containing ribose as the sugar component and which, most generally, is transcribed (copied) from DNA. RNA molecules may serve informational (e.g., messenger RNA), catalytic (e.g. RNase P), or structural (e.g., ribosomal RNA, see below) cellular functions.
rRNA--Ribosomal RNA (rRNA) molecules are key elements of ribosomes, complex protein and RNA-containing "organelles" which, together with transfer RNAs, comprise the translation apparatus. Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, in E. coli, are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by sedimentation rate. However, they actually vary substantially in size between organisms. 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and will be so used here. The evolutionary homologs of these three RNA molecules are present in the ribosomes of all organisms. In eukaryotes, the homologous RNAs are designated 5S, 18S, and 28S rRNA, respecively. Eukaryotes contain, in addition, a fourth rRNA species named 5.8S rRNA whose structural and, presumably, functional homolog is found at the 5' end of bacterial 23S rRNA.
Hybridization--the process by which, under defined reaction conditions, two partially or completely complementary nucleic acids are allowed to come together in an antiparallel fashion and form specific and stable hydrogen bonds. The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization.
Probe(s)--synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize, under defined stringencies, specifically (i.e., preferentially) to target nucleic acid sequences.
Target molecule--a nucleic acid molecule to which a particular probe is capable of preferentially hybridizing.
Target sequence--a nucleic acid sequence within the target molecules to which a particular probe is capable of preferentially hybridizing.
Salmonella-specific sequences--nucleotide sequences within a target molecule which exhibit significant sequence differences between Salmonella and non-Salmonella enteric bacteria.
Other definitions are given as their first use arises in the text.